title: Improving molecular tools for global surveillance of measles virus.
abstract: BACKGROUND
  The genetic characterization of wild-type measles viruses plays an important role in the description of viral transmission pathways and the verification of measles elimination. The 450 nucleotides that encode the carboxyl-terminus of the nucleoprotein (N-450) are routinely sequenced for genotype analysis.
  
  
  OBJECTIVE
  The objectives of this study were to develop improved primers and controls for RT-PCR reactions used for genotyping of measles samples and to develop a method to provide a convenient, safe, and inexpensive means to distribute measles RNA for RT-PCR assays and practice panels.
  
  
  METHODS
  A newly designed, genetically defined synthetic RNA and RNA isolated from cells infected with currently circulating genotypes were used to compare the sensitivity of primer pairs in RT-PCR and nested PCR. FTA® cards loaded with lysates of measles infected cells were tested for their ability to preserve viral RNA and destroy virus infectivity.
  
  
  RESULTS
  A new primer pair, MeV216/MeV214, was able to amplify N-450 from viruses representing 10 currently circulating genotypes and a genotype A vaccine strain and demonstrated 100-fold increased sensitivity compared to the previously used primer set. A nested PCR assay further increased the sensitivity of detection from patient samples. A synthetic positive control RNA was developed that produced PCR products that are distinguishable by size from PCR products amplified from clinical samples. FTA® cards completely inactivated measles virus and stabilized RNA for at least six months.
  
  
  CONCLUSIONS
  These improved molecular tools will advance molecular characterization of circulating measles viruses globally and provide enhanced quality control measures.
authors.affiliation: Measles, Mumps, Rubella and Herpes Virus Laboratory Branch, Division of Viral Diseases, Centers for Disease Control and Prevention, MS C-22, 1600 Clifton Road, Atlanta, GA 30333, USA. bbankamp@cdc.gov
authors.forename: Bettina
authors.initials: B
authors.lastname: Bankamp
authors.forename: Lauren A
authors.initials: LA
authors.lastname: Byrd-Leotis
authors.forename: Elena N
authors.initials: EN
authors.lastname: Lopareva
authors.forename: Gibson K S
authors.initials: GK
authors.lastname: Woo
authors.forename: Chunyu
authors.initials: C
authors.lastname: Liu
authors.forename: Youngmee
authors.initials: Y
authors.lastname: Jee
authors.forename: Hinda
authors.initials: H
authors.lastname: Ahmed
authors.forename: Wilina W
authors.initials: WW
authors.lastname: Lim
authors.forename: Nalini
authors.initials: N
authors.lastname: Ramamurty
authors.forename: Mick N
authors.initials: MN
authors.lastname: Mulders
authors.forename: David
authors.initials: D
authors.lastname: Featherstone
authors.forename: William J
authors.initials: WJ
authors.lastname: Bellini
authors.forename: Paul A
authors.initials: PA
authors.lastname: Rota
mesh_terms: D017931:DNA Primers / Q000235:genetics; D062665:Epidemiological Monitoring; D005838:Genotype; D014943:Global Health; D006801:Humans; D008457:Measles / Q000175:diagnosis* / Q000453:epidemiology* / Q000821:virology; D008459:Measles virus / Q000145:classification / Q000235:genetics* / Q000302:isolation & purification*; D025202:Molecular Diagnostic Techniques / Q000379:methods* / Q000592:standards; D017720:Molecular Epidemiology / Q000379:methods; D012367:RNA, Viral / Q000235:genetics; D012015:Reference Standards; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods / Q000592:standards; D012680:Sensitivity and Specificity
chemical_list: D017931:DNA Primers; D012367:RNA, Viral
keywords: CDC; Centers for Disease Control and Prevention; Ct; FTA(®) cards; Genotyping; LabNet; MeV; Measles; Molecular diagnostics; RT; WHO; WHO Measles and Rubella Laboratory Network; World Health Organization; cpe; cytopathic effect; measles virus; nt; nucleotides; room temperature; threshold cycle